An injectable gelatin/sericin hydrogel loaded with human umbilical cord mesenchymal stem cells for the treatment of uterine injury

Abstract Abnormal endometrial receptivity is a major cause of the failure of embryo transplantation, which may lead to infertility, adverse pregnancy, and neonatal outcomes. While hormonal treatment has dramatically improved the fertility outcomes in women with endometriosis, a substantial unmet need persists in the treatment. In this study, methacrylate gelatin (GelMA) and methacrylate sericin (SerMA) hydrogel with human umbilical cord mesenchymal stem cells (HUMSC) encapsulation was designed for facilitating endometrial regeneration and fertility restoration through in situ injection. The presented GelMA/10%SerMA hydrogel showed appropriate swelling ratio, good mechanical properties, and degradation stability. In vitro cell experiments showed that the prepared hydrogels had excellent biocompatibility and cell encapsulation ability of HUMSC. Further in vivo experiments demonstrated that GelMA/SerMA@HUMSC hydrogel could increase the thickness of endometrium and improve the endometrial interstitial fibrosis. Moreover, regenerated endometrial tissue was more receptive to transfer embryos. Summary, we believed that GelMA/SerMA@HUMSC hydrogel will hold tremendous promise to repair or regenerate damaged endometrium.


| INTRODUCTION
The uterus is one of the most important reproductive organs for women, and the endometrium (as the place for implantation, embryonic development, and pregnancy maintenance of female fertilized eggs) is the key factor for the success of pregnancy. 1,2 However, endometrial damage was caused by physical injury (induced abortion, frequent uterine surgery) and biochemical injury (infection, endocrine disrupting chemicals), which could lead to impaired endometrial proliferation and affect embryo implantation and implantation, resulting in female infertility or repeated abortion. [3][4][5] Currently, there are still big challenges for promoting the repair and regeneration of endometrium after injury. For the regeneration and repair of endometrial injury, the traditional treatment mainly includes surgical treatment, intrauterine barrier, endocrine therapy, increasing endometrial blood perfusion, and so on. 6,7 However, the methods to promote endometrial regeneration after the moderate and severe injury are still very limited, which could not avoid endometrial regeneration disturbance and postoperative adhesion recurrence. Therefore, the key to successful treatment is the ability to prevent the reformation of uterine adhesion and promote the regeneration and repair of endometrium.
Stem cells have the pluripotency, the ability to release growth factors and regulate inflammation, which showed great potential in the treatment of a variety of injuries and diseases in regenerative medicine. [8][9][10] Mesenchymal stem cells (MSCs, a kind of adult stem cells), which have the ability of stem cell proliferation and multidirectional differentiation, which could be isolated from a variety of tissues (such as umbilical cord, endometrial polyps, menstrual blood, bone marrow, adipose tissue, etc). [11][12][13] Currently, a large number of animal experiments showed that MSC could improve the repair of endometrial injury and increase the pregnancy rate. 14,15 In the mouse model of endometrial injury, intraperitoneal transplantation of endometrial mesenchymal stem cells (EnMSC) could effectively repair the damaged endometrium, increase the uterine microangiogenesis, and improve the pregnancy rate. 16 Prior study proposed that intravenous injection of human umbilical cord mesenchymal stem cells (HUMSC) could increase the thickness and glands of endometrium, increase the implantation rate of embryos, reduce the excessive fibrosis, promote the vascular growth and endothelial cell proliferation, regulate the inflammatory factors, and restore the structure and function of ethanol-damaged endometrium. 3 Moreover, HUMSC have the characteristics of low immunogenicity, high ability of self-replication, noninvasive collection, which could promote the regeneration and repair of injured tissue. However, there are still some shortcomings in the clinical application of MSC therapy, such as the tumorigenic potential of stem cells, thrombosis, fever, and other adverse reactions. 17,18 Meanwhile, the low efficiency of cell implantation caused by local injection greatly limited the clinical promotion of stem cell therapy. 19 One of the main reason for the low cell retention rate is the lack of three-dimensional matrix to support the survival, migration, and development of transplanted cells. 20 To resolve these problems, a variety of injectable hydrogel systems have been developed, which could provide mechanical protection to prevent cell membrane from being destroyed during injection and form a stable network after injection, resulting in promoting cell adhesion and growth. [21][22][23][24][25] However, many available injectable hydrogels have some problems such as poor mechanical properties, low cell survival rate, or inability to accurately control their gelation process and gel properties. 26,27 Prior study reported that the natural extracellular matrix (ECM) environment played a key role in influencing through a series of complex physical, mechanical, and biochemical signals. 28,29 To further understand the regulation of cell behavior by ECM, designing new materials with precise adjustable structure, mechanical properties, biodegradability, and cell interaction has been a challenging task for the research community. Therefore, our goal is to design an injectable and adjustable hydrogel crosslinked by blue or ultraviolet light to meet the different requirements of cell culture and tissue engineering applications.
Gelatin (Gel) is a derivative of collagen, which is often applied in hydrogels because of its good gelling, biocompatibility, and biodegradability. 30 However, one limitation of gelatin-based hydrogels is poor mechanical properties and thermal stability. 31 These limitations can be overcome by chemical or physical cross-linking of gelatin. In addition, the addition of other ECM components can enrich gelatin hydrogels. Sericin (Ser) is a main component of natural silk and has good water solubility and excellent biocompatibility. 32,33 Meanwhile, sericin can promote cell adhesion and proliferation, antioxidant, and inhibit tyrosinase activity. 34 These advantages make sericin gradually become the research focus of new natural materials in the field of tissue engineering and regenerative medicine. 35 In addition, the interpenetrating polymer network (IPN) hydrogel is a unique structure, which allows two independent networks to combine with each other (maintain the required properties of the original polymer) to obtain better mechanical properties. 36,37 In this study, we hypothesized that the modified gelatin and sericin could effectively cross-link into IPN hydrogel structure, which is suitable for HUMSC cells to be incorporated into the injured tissue and play a role in tissue repair.
In this study, we aim to develop an injectable hydrogel based on methacrylate gelatin (GelMA) and methacrylate sericin (SerMA) matrix as cell delivery carriers of HUMSC to promote endometrial repair (Scheme 1). The porous structure, swelling properties, mechanical properties, and degradation properties of this hydrogel were characterized. In addition, the cytocompatibility of hydrogels was studied in detail by CCK-8 and living/dead cell staining using the L929 and HUMSC cells. Meanwhile, cell encapsulation ability was characterized by three-dimensional live dead staining and cytoskeleton staining. And excellent therapeutic effect for promoting the endometrial repair and fertility restoration were shown in vivo experiments. In summary, all of these results demonstrated that the GelMA/ SerMA@HUMSC hydrogel will have great potential in the repair of endometrial injury.

| Synthesis of methacrylated gelatin (GelMA)
The preparation of GelMA follows the reported method in the literature with slight modification. 38 Briefly, 10 g of gelatin was dissolved 100 ml of 50 C preheated deionized water. Then, 6 ml of MA reagent was slowly dripped into the gelatin solution, the final solution was stirred for 12 h. The reaction was performed in darkness, a white milky solution was obtained after the completion of the reaction. The obtained solution was centrifuged for at 6000 rpm for 5 min and the supernatant was dialyzed in a dialysis bag (14-kDa molecular weight S C H E M E 1 (a) Preparation of GelMA/SerMA hydrogel. (b) GelMA/SerMA hydrogel system encapsulating HUMSC for the treatment of uterine injury by injection into the uterine cavity cutoff) against distilled water at 40 C for 6 days. Finally, the solution was freeze-dried to obtain white porous foam-like GelMA prepolymer and stored it in a refrigerator at À20 C for later use.

| Synthesis of methacrylated sericin (SerMA)
Sericin was extracted from natural silkworm cocoon through the high heat/alkaline degumming method according to a previously reported procedure with minor modifications. 39 Briefly, 40 g of cocoons were cut into small pieces and boiled in Na 2 CO 3 solution (1 L, 0.02 M) for 1 h. Then, the insoluble residue was removed by miracloth and the resultant solution was dialyzed (MWCO, 3500 Da) against distilled water for 3 days. Sericin was then obtained by lyophilization.
SerMA was synthesized following previously published protocols. 40 A 10 g of sericin was dissolved 100 ml of deionized water. Then, 6 ml of MA reagent was slowly dripped into the sericin solution and the pH was adjusted to 9.0 with 2 M NaOH during the reaction. After stirring for 1 h at room temperature, the pH of the solution was stabilized at 7.0 using 1 M HCl. Then, the insoluble residue was removed by miracloth and the resultant solution was dialyzed (MWCO, 3500 Da) against distilled water for 3 days. Finally, SerMA are obtained by lyophilization and stored it in a refrigerator at À20 C for later use.

| Preparation of GelMA/SerMA hydrogel
The concentration of GelMA was fixed at 20% (w/v) in deionized water, and varying amounts of SerMA (5%, 10%, and 15% [w/v]) were dissolved in deionized water. The two solutions were then mixed at a volume ratio 1:1, and LAP was added at 0.1% (w/v). The mixtures were exposed to a UV light (365 nm) at a power density of 5 mW/cm 2 for 30 s to produce GelMA/SerMA hydrogels.

| Characterization of polymers
The chemical structure of GelMA and SerMA was characterized by 1 H NMR (Bruker 400 MHz NMR spectrometer). The chemical construction of GelMA and SerMA was characterized by Fourier transform infrared spectroscopy (FTIR; ThermoScientific, Waltham, USA). The micromorphology of GelMA and GelMA/SerMA hydrogels was observed by scanning electron microscopy (SEM; S-3400, Hitachi, Japan) with an accelerating voltage of 5 kV. The averaged pore diameter of was calculated using Nano measure software.

| Swelling ratio
The swelling ratio of the composite hydrogel was investigated using a quality method. 41 Briefly, 400 μl of hydrogel was immersed in phosphate buffered solution (PBS) (2 ml, pH 7.4) and incubated at 37 C in an incubator. At indicated time points (2,4,6,8, and 24 h), the hydrogel was taken out and weighted after wiping off all surface water with a filter paper.
The swelling ratio (%) was calculated based on the following formula: where W 0 represents the initial weight of the hydrogel and W t means the weight of the swollen hydrogel at time t.

| Rheological tests
Rheological measurements of the composite hydrogel were performed using a rotated rheometer (AR 2000ex; TA Instrument, USA).
In the time testing, the changes of the storage modulus (G') and loss modulus (G") were registered over time with the frequency of 1 Hz and the shear strain of 1%. In the frequency testing, the range of frequency was 0.1-10 Hz with a shear strain of 1%.

| Compression test
Compression experiments were conducted on a universal testing machine (Instron5543A; Boston, USA). A volume of 600 μl of hydrogel solutions were cured for 30 s in a 48-well plate and placed at the center of the lower compression plate. The compression rate of 1 mm/ min was applied, and strain level was up to ≈40% of the original height.

| In vitro biodegradation
The degradation of hydrogels in vitro was measured following the method reported in the literature with slight modification. 42

| Histological examination
The uterine tissues were fixed in 4% paraformaldehyde solution overnight, followed by dimethylbenzene, and embedded in paraffin. All tissue blocks were cut into serial 4 μm thick sections and stained with stained with hematoxylin and eosin (H&E) and Masson's trichrome staining. Immunohistochemistry of TGF-β1 was performed according to the previous steps. 45 The pictures of the stained sections were captured using a microscope (RM2016; Leica).

| Immunofluorescence staining
After paraffin sections rehydrated, 4 μm-thick sections were blocked using 5% BSA, after which they were incubated with mouse anti-CD31 (Servicebio, GB13428, 1:1000), mouse anti-CK18 (Servicebio, Membranes were then washed three times with TBST and incubated with the HRP-conjugated anti secondary antibody. The ImageJ software was used for densitometric analyses of protein bands.

| Fertility evaluation
The fertility of each group of mice was evaluated after three estrous cycles. Briefly, the female mice naturally mated with male mice in cages, and the ratio of female-to-male mice was 2:1. At 16 days after the occurrence of vaginal suppository in female mice, the female mice were sacrificed to confirm whether they were pregnant or not.

| Statistical analysis
All data were expressed as the average value ± standard deviation at

| Swelling ratio assay
As shown in Figure 2a, the swelling degree of GelMA and GelMA/ SerMA hydrogels increases with time and is stable in PBS from 8 to 24 h. All hydrogels became almost saturated after 8 h, and reach their equilibrium swelling. The swelling ratio of SerMA-free GelMA, GelMA/10%SerMA, GelMA/15%HAMA, and GelMA/20%HAMA hydrogels were 22.8% ± 0.6%, 24.5% ± 1.1%, 24.8% ± 0.9%, and 27.1% ± 0.7%, respectively. These data showed that the prepared hydrogel will not expand due to absorbing too much water in the process of tissue application in vivo, thus avoiding the damage to the surrounding tissue. The addition of SerMA slightly increased the swelling rate of the hydrogel, which may be due to more holes in GelMA/ SerMA hydrogel. Figure 2b shows the variation curve of the storage modulus (G') and the loss modulus (G") of hydrogels with time. It can be seen that both G' and G" do not change significantly with time, and G' has always been greater than G", indicating that the hydrogel has good stability.

| Rheological and compression analysis
Meanwhile, the G' of the GelMA/SerMA hydrogel is higher than that of GelMA hydrogels, indicating that the stiffness of the GelMA/ SerMA hydrogel is larger than that of GelMA hydrogels. Figure 2c showed the relationship between the G' and the G" of the hydrogel at the rotational speed of the rheometer tray at 0.1-10 Hz. It can be found that the G' of the four kinds of hydrogel materials is larger than G", which indicates that the hydrogel can stably maintain its solid state at this frequency.
An ideal hydrogel for endometrial repair should equip with good mechanical properties to keep its integrity during use. 46 Figure 2d showed the test results of Young's compression modulus of hydrogel material under 40% strain. It can be found that GelMA/20%SerMA hydrogel has the largest compression modulus (118 kPa), which showed strong rigidity. Notably, the compression modulus of GelMA/10%SerMA was 40 kPa, which was more suitable for application of uterine injury and close to that of human tissues and organs. 47

| In vitro degradation
Degradation of biomaterial is one of the most important properties regarding their application in biology, which are directly related to their service life. 48 The weights of GelMA and GelMA/10%SerMA hydrogels were gradually decreased with the incubation time ( Figure 3a,b). Compared to the samples of GelMA hydrogel, the samples of GelMA/10%SerMA hydrogel showed a low weight loss rate, which might due to the higher cross-linking strength. The same degradation trend also appeared in lysozyme conditions. The hydrogels were rapidly degraded when the samples were subjected to lysozyme solutions. This might be because the gelatin and sericin chains were decomposes by lysozyme. 49 These results indicated that GelMA/10%SerMA hydrogel exhibited a stable degradation rate and gradually degraded within 4 weeks, which will provide a favorable property for its application in vivo. Meanwhile, the micromorphology of freeze-dried GelMA and GelMA/10%SerMA hydrogels after degradation was shown in

| Biocompatible of hydrogel
In order to study the biocompatibility of composite hydrogel, we evaluated the survival ability and proliferation effect of L929 and HUMSC it can be seen that L929 and HUMSC cells showed good growth and adhesion on GelMA and GelMA/SerMA hydrogels. After 3 and 5 days of culture, the survival rate of L929 and HUMSC cells in each group was higher than 90% (Figure 4b,d), and there was no significant difference (p > 0.05). As shown in Figure S2, the number of HUMSC cells on the GelMA/SerMA hydrogel increased with incubation time.
These data showed that the prepared hydrogels have good biocompatibility and cells can adhere and grow better on GelMA/SerMA hydrogels, which could be used as a biomaterial carrying cells.
The effect of GelMA and GelMA/SerMA on HUMSC cell migration was evaluated (Figure 4e). The culture medium supplemented with the same volume of PBS was used as control. For HUMSC cells, GelMA/SerMA group showed a significant increase at 4 h when compared to the control group. After incubation for 8 h, cells exposed to GelMA/SerMA group showed the highest migration (98.0% ± 0.2%), followed by those exposed to GelMA group (93.2% ± 2.3%) and control group (86.8% ± 1.0%). The increased cell migration rates for cells

| Cell encapsulation in hydrogel
To further evaluate whether the GelMA and GelMA/SerMA hydrogels can be served effectively as cell carriers, the encapsulation, and culture of HUMSC within hydrogels were performed. After 1, 4, and 7 days of incubation, the overall distribution of cells in hydrogels was uniform and most of the cells were alive (Figure 5a), suggesting that HUMSC cells could survive in the 3D encapsulation process. Next, the proliferation of HUMSC cells in the hydrogel was measured by CCK-8 method. Figure 5b showed that after the GelMA/SerMA hydrogel encapsulated with HUMSC cells was cultured for 1 day, the optical density (OD) value was 0.55, OD increased to 1.19 for 4 days, and the OD value continued to rise to 1.75 for 7 days. The higher OD value was due to that the gelatin and sericin materials in hydrogel are important ECM, have excellent biocompatibility and contain specific sites that bind to cells, which can promote the adhesion and proliferation of cells. As shown in Figure 5c, the morphology of HUMSC grown on GelMA/SerMA hydrogel was different from that on GelMA hydrogel, which exhibited more elongation and thicker actin filaments.
These results suggested that SerMA, as natural polymers, provided a favorable microenvironment for cell adhesion and proliferation, demonstrating that this functionalized sericin is a good candidate material for tissue engineering.

| In vivo analyses of endometrial thickness
Next, we established a mice model of endometrial injury through directly injecting 95% ethanol into the uterine horn. After modeling, the mental state of the mice in the model group and the sham operation group was normal, the diet was good and the estrous cycle was regular. As shown in Figure S3,  CD31 is an important index to evaluate neovascularization. 51 As shown in Figure 7a, the distribution of CD31 in the sham operation group was more than that in the model group, while the expression of CD31 in the GelMA/SerMA hydrogel treatment group was between the sham operation group and the model group. As shown in Figure S5A, the CD31 positive ratio of sham operation group, model group, GelMA/SerMA hydrogel group, and GelMA/SerMA@HUMSC hydrogel group were 8.2% ± 1.0%, 15.4% ± 1.4%, 13.8% ± 0.6%, and 23.9% ± 2.5%, respectively. CK18 is member of the keratin family of intermediate filament proteins, which is more expressed in the cytoplasm of endometrial epithelial cells. 52 As shown in Figure S5B, the expression of CK18 in the sham operation group is more than that in the model group, while that in the model group is less. The expression of CK18 in the GelMA/SerMA hydrogel treatment group and the GelMA/SerMA@HUMSC hydrogel treatment group is more than that in the model group, indicating that the efficacy in the hydrogel treated group is better than that in the model group.

| Immunofluorescence staining
Ki67 is an important index to evaluate cell proliferation. 53 There is more proliferation in the sham operation group and less in the model group. As shown in Figure S5C, the expression of Ki67 in the GelMA/ SerMA hydrogel treatment group and the GelMA/SerMA@HUMSC hydrogel treatment group is more than that in the model group, indicating better cell proliferation and tissue growth. Vimentin is an indicator of the specific expression of Vimentin in endometrial stromal cells. 54 As shown in Figure S5D, the expression of CK18 in the sham operation group is more than that in the model group, while the expression of CK18 in the GelMA/SerMA hydrogel treatment group and the GelMA/ SerMA@HUMSC hydrogel treatment group is more, and the expression effect is the same as that in the sham operation group, indicating that the combination of hydrogels with HUMSC may achieve better therapeutic effect. Caspase-3 activation is a hallmark of apoptotic cell death.
As shown in Figure S5E, the expression of Caspase 3 in GelMA/SerMA hydrogel treatment group and the GelMA/SerMA@HUMSC hydrogel treatment group. As shown in Figure 7b,c, western blot analysis showed a similar trend.

| Fertility evaluation
To study the effect of hydrogel transplantation on the fertility of mice with endometrial injury, hydrogel was transplanted into the uterine cavity of mice after modeling, and female mice and male mice were mated in cages after three estrous cycles. As shown in Figure

CONFLICT OF INTERESTS
The authors declare no competing financial interest.

DATA AVAILABILITY STATEMENT
Some or all data, models, or code generated or used during the study are available from the corresponding author by request.